γ-Synuclein: Seeding of α-Synuclein Aggregation and Transmission between Cells

γ-Synuclein Seeding of α-Synuclein Aggregation and Transmissionbetween CellsIrina SurguchevadaggerDagger Victor S Sharovsect and Andrei SurguchovdaggerDagger

daggerRetinal Biology Laboratory Veterans Administration Medical Center 4801 East Linwood Boulevard Kansas City Missouri 66148United StatesDaggerDepartment of Neurology Kansas University Medical Center Kansas City Kansas 66160 United StatessectDepartment of Pharmaceutical Chemistry University of Kansas Lawrence Kansas 66045 United States

ABSTRACT Protein misfolding and aggregation is a ubiquitous phenomenonassociated with a wide range of diseases The synuclein family comprises three smallnaturally unfolded proteins implicated in neurodegenerative diseases and some formsof cancer α-Synuclein is a soluble protein that forms toxic inclusions associated withParkinsonrsquos disease and several other synucleinopathies However the triggersinducing its conversion into noxious species are elusive Here we show that anothermember of the family γ-synuclein can be easily oxidized and form annular oligomersthat accumulate in cells in the form of deposits Importantly oxidized γ-synuclein caninitiate α-synuclein aggregation Two amino acid residues in γ-synuclein methionineand tyrosine located in neighboring positions (Met38 and Tyr39) are most easilyoxidized Their oxidation plays a key role in the ability of γ-synuclein to aggregate andseed the aggregation of α-synuclein γ-Synuclein secreted from neuronal cells intoconditioned medium in the form of exosomes can be transmitted to glial cells andcause the aggregation of intracellular proteins Our data suggest that post-translationally modified γ-synuclein possesses prion-likeproperties and may induce a cascade of events leading to synucleinopathies

Protein misfolding and aggregation is one of the mostexciting new frontiers in protein chemistry and molecular

medicine These processes may be associated with a gain oftoxic function and a loss of physiological function Abnormalaggregation of neuronal proteins plays a key role in thepathogenesis of many neurodegenerative diseases (NDDs)1minus5

Parkinsonrsquos disease (PD) is characterized by the loss of thedopaminergic neurons and the deposition of α-synuclein-containing Lewy bodies4minus6 α-Synuclein aggregation anddopamine (DA) metabolism are linked to PD pathogenesis78

Neurotransmitter DA and its derivatives promote accumulationof oligomeric or protofibril forms of α-synuclein910

Another property of α-synuclein that is associated with itsrole in pathology in addition to its propensity to aggregate is itsability to be secreted11minus15 Furthermore the transmission ofaggregated α-synuclein between cells might be considered as amechanism of transmission of pathology16

Both monomeric and oligomeric α-synuclein forms aresecreted from differentiated human neuroblastoma cells andprimary cortical neurons17 Extracellular α-synuclein aggregatesare neurotoxic for any cells exposed to them Recent datasuggest that the mechanism of internalization of extracellular α-synuclein is dependent on the assembly state of the protein18

Transmission of α-synuclein from neurons to astroglia causesthe formation of inclusion bodies and alterations in the patternof gene expression consistent with the inflammatoryresponse19 This transmission of α-synuclein might be animportant mediator of pathogenic glial responses and therefore

may be considered as a new therapeutic target Exogenouslyadded to cell culture medium recombinant α-synuclein isinternalized by the recipient cells1820minus22 leading to celldeath23minus25

The interaction with recombinant α-synuclein can activatemicroglial cells and cause neurotoxicity192526 In addition α-synuclein aggregates can propagate pathology via neuron-to-neuron transmission27minus29 The mechanism of secretion andendocytosis may play an important role in the pathogenesis ofsynucleinopathies because intercellular cell-to-cell transfer of α-synuclein causes aggregation of α-synuclein in recipientneurons29 Thus pathogenic actions of α-synuclein extend tothe extracellular space and neighboring cells30

Aggregational properties and the mechanism of secretion andendocytosis are investigated primarily for α-synuclein29minus31

whereas much less is known about two other members of thesynuclein family β- and γ-synuclein (Figure 1A reviewed in ref3) γ-Synuclein like α-synuclein is able to form aggregates andfilaments both in vitro32 and in vivo33 Such γ-synucleininclusions are found in histopathological lesions in severalreports of human pathology33minus36 and in transgenic mod-els37minus39

Because γ-synuclein is considered a causative factor in thedevelopment of certain neurological disorders because of its

Received April 13 2012Revised May 22 2012Published May 23 2012

Article

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copy 2012 American Chemical Society 4743 dxdoiorg101021bi300478w | Biochemistry 2012 51 4743minus4754

aberrant accumulation these disorders may be termed γ-synucleinopathies3338

Here we show that γ-synuclein can seed α-synucleinaggregation this seeding is especially efficient after γ-synucleinoxidation Furthermore γ-synuclein can be secreted fromneuronal cell cultures and internalized by glial cells Theseprocesses might have an important role in the development andspreading of synucleinopathies

EXPERIMENTAL PROCEDURES

Antibodies (Abs) for WB and ImmunofluorescenceThe following Abs were purchased from Santa CruzBiotechnology Inc (Santa Cruz CA) α-synuclein-specificantibody [sc-7011-R (C-20)] and CD63-specific Ab (H-193)(sc-15363) β-Synuclein anti-rabbit polyclonal Ab AB5068 wasobtained from Chemicon (Temecula CA) γ-Synuclein Absused for WB were either anti-rabbit (Ab6169) or anti-mouse(1H10D2) clones from ABcam (Cambridge MA) Mousemonoclonal Ab 2C3 against γ-synuclein (GenTex) was used forimmunofluorescence The Ser129-phosphorylated α-synucleinwas revealed with anti-mouse monoclonal Ab from Wacowhich recognizes predominantly aggregated α-synuclein21

Lectin-PHA-L-conjugated by Alexa488 (InvitrogenMolecularProbes Eugene OR) was used as a membrane marker and anti-mouse Ab against GM-130 [Becton Dickinson (BD) FranklinLakes NJ] as a Golgi marker Secondary Abs (goat anti-mouseAlexa488 and Qdot-655 anti-rabbit) were purchased fromInvitrogenMolecular Probes Anti-rabbit Cy3-conjugated Abwas from Jackson ImmunoResearch Lab Inc (West GrovePA)Generation of Ab to γ-Synuclein with an Oxidized

Methionine 38 (Met38) (21st Century BiochemicalsMarlboro MA) Ab was raised in rabbits using as an antigenpeptide Ac-TKEGV[M-O]YVGAKT-Ahx-C-amide where [M-O] is methionine sulfoxide The peptide was purified by high-performance liquid chromatography and its sequence wasconfirmed by MSMS Nanospray analysis After the fourthbleeding the Ab was affinity purified on a column containingantigenic peptide Purified Ab was immunodepleted using animmunodepletion column with unmodified peptide withoutoxidized methionine (acetyl-TKEGVMYVGAKT-Ahx-C-amide) For Western blotting (WB) the Ab was used at a1500 dilutionAnalysis of α- β- and γ-Synucleins Colloidal

Coomassie Brilliant Blue Staining The protein samples wereseparated in a 12 gel and fixed in 125 TCA for 1 h The gelwas stained with 01 G-250 brilliant blue in 20 methanolcontaining 10 (NH 4)2SO4 and 2 H3PO4 overnight at RTand washed with H2OAggregation Assays In Vitro Assay Recombinant α- β-

and γ-synucleins were from ABcam (α-synuclein ab48842 β-synuclein ab48853 and γ-synuclein ab48712 respectively) Theproteins were diluted by PBS to 05 μM mixed with a 20-foldexcess of DA and incubated overnight at 37 degC The sampleswere boiled for 3 min in a sample buffer (SB) with 5 β-mercaptoethanol After the treatment we assessed theformation of SDS-resistant protein aggregates by electro-phoresis in a 12 or 4 to 15 PAAGAssay of Cell Extracts A7 cells were split in a 96-well plate

in the regular medium The next day γ-synuclein preincubatedwith DA or PBS buffer was added to the cells The cells werewashed with PBS after different periods of incubation (0 6 24

and 48 h) and suspended in 2times SB The samples were boiledand analyzed in a 4 to 15 PAAG in the presence of SDS

Incubation with DA or L-DOPA α- β- and γ-synucleinswere incubated overnight with a 20-fold molar excess of DA inPBS Because of the low stability of DA in experiments withlonger periods of incubation it was replaced with a similarconcentration of a more stable DA precursor 34-dihydrox-yphenyl-L-alanine (L-DOPA)

Cell Cultures The immortalized primary culture of ratoptic nerve A7 astrocytes was a generous gift from H Geller(National Institutes of Health Bethesda MD)40 The culturewas immortalized with murine leukemia virus psi-2 SV-40-6which is defective in assembly and contains the SV-40 large Tantigen and neomycin-resistance genes as described previ-ously40 This stable immortalized clonal cell line expressednuclear SV-40 large T cells and the astrocyte-specific markerglial fibrillary acidic protein (GFAP) The cells were grown asdescribed previously41minus43

Human neuroblastoma cell line SH-SY5Y was purchasedfrom ATCC (catalog no CRL-2266) and cultivated in DMEMF-12 medium supplemented with 10 fetal bovine serum at 37degC in 5 CO2 Astrocytoma grade IVhigh-grade astrocytoma(U-87 MG) (ATCC catalog no HTB-14) was cultivated inEaglersquos Minimal Essential Medium with Earlrsquos BSS and 2 mMglutamine (EMEM) adjusted to contain 10 mM sodiumpyruvate 01 mM nonessential amino acids and 15 gLsodium bicarbonate (90) and fetal bovine serum (10)Other details are described by the manufacturer and in ourprevious publication42

Immunofluorescent Staining SH-SY5Y Cells They weresplit on coverslips covered with poly-D-lysine The cells werewashed with cold PBS and stained with PHA-L conjugated withAlexa488 for 15 min at 4 degC before being fixed (4 PFA for 15min at RT) After being fixed the cells were washed andblocked in 3 BSA 3 NGS and 01 Triton X-100 in PBSfor 2 h at RT The coverslips were incubated with the primaryAb overnight at 4 degC After being stained with the secondary Abfor 1 h at RT the coverslips were put in a mounting solutioncontaining DAPI-Vectashield (Vector Laboratories Burlin-game CA) Other details are described in ref 44

U-87 MG Cells They were split on the coverslips in a 12-wellplate in the regular medium The next day the medium wasreplaced for the CMs from the stable clones produced in SH-SY5Y cells The cells were grown for 7 or 12 days at 37 degC inthe presence of 5 CO2 Before being fixed the cells werewashed with warm PBS containing Ca2+ and Mg2+ and thenfixed with 4 PFA for 15 min at RT The cells were washedwith PBS permeabilized with 03 Triton X-100 (15 min atRT) and blocked in a solution containing 3 BSA 3 NGSand 01 Triton X-100 in PBS for 2 h at RT The coverslipswere placed in the primary antibody overnight at 4 degC Afterbeing stained with the secondary antibody for 1 h at RT thecoverslips were placed in a mounting solution containingDAPI-Vectashield (Vector Laboratories) Nonfixed cells werewashed with PBS and stained with PHA-L conjugated withAlexa488 for 2 h at RT before being fixed with 4 PFA for 15min at RT

Confocal Microscopy The images of U-87 cells weretaken with a Nikon TE 2000 Inverted 3 Laser confocalmicroscope driven by Nikon EZ-C1 391 software Thefollowing lasers were used 488 543 and 633 nm Nikon EZ-C1Free Viewer software was used for the reconstruction oforthogonal projections

Biochemistry Article

dxdoiorg101021bi300478w | Biochemistry 2012 51 4743minus47544744

Expression of γ-Synuclein in SH-SY5Y Cells For thegeneration of γ-synuclein-overexpressing clones pcINeoplasmid-containing human γ-synuclein cDNA was transfectedinto SH-SY5Y cells using Lipofectamine 2000 (InvitrogenMolecular Probes) The clones resistant to antibiotic G-418(300 μgmL) were selected For the generation of γ-synucleinand mutant γ-synuclein fused with GFP clones the pEGFP-N1vector was used as a template for amplification with primerscontaining restriction site recognition sequences for cloning(Clontech Mountain View CA) The following primers wereused 5prime-ATC TCG AGA TGG ATG TCT TCA-3prime (forward)(with the XhoI recognition site) and 5prime-TTA AGC TTG TCTCCC CCA CTC-3prime (reverse) (with the HindIII site)Amplification was conducted with a kit for GC-rich sequences(Roche) and the generated polymerase chain reaction productwas cloned into the XhoI and HindIII sites of the pEGFP-N1vector γ-Synuclein-GFP mutant clones were generated by thesame procedure the exception being that pTrc-His6-γ-synuclein constructs that already had mutations in cDNAswere used for the expression of proteins in Escherichia coliOverexpression of wt and A53T Mutant α-Synuclein

SH-SY5Y cells overexpressing wild-type (wt) and A53T mutantα-synuclein were generated using lentivirus constructs asdescribed previously27 (generous gift of E Masliah) In oneset of experiments the stable clones overexpressing wt-γ-synuclein fused with GFP were infected with the lentivirusconstruct carrying wt-α-synuclein The cells were grown for 48h in the regular medium containing 300 μgmL antibiotic G-418 Extracts were analyzed by WB with anti-rabbit Ab C-20(Santa Cruz Biotechnology Inc) specific for α-synucleinDelivery of γ-Synuclein into the SH-SY5Y Cell Line by

BioPorter Cells were split in a 24-well plate in the regularmedium γ-Synuclein was diluted with PBS to 01 mgmLtreated by 1 mM DA and incubated overnight at 37 degCProtein samples were adjusted to 40 μL by PBS and transferredto tubes with lyophilized BioPorter (Sigma) Subsequentprocedures were conducted as recommended by themanufacturer Before the mixtures with BioPorter were addedto the cells the regular medium was replaced with SFM(Invitrogen Carlsbad CA) The cells were grown for 24 h theserum was added to a final concentration of 10 and thegrowth was continued for an additional 24 h FITC-IgG protein(Sigma) was used as a controlSecretion of γ-Synuclein from SH-SY5Y Cells SH-SY5Y

stable clones overexpressing γ-synuclein or γ-synuclein-pGFPwere grown for 24 h in the presence of 10 FBS and themedium was replaced with SFM (GibcoLife TechnologiesGrand Island NY) After 48 h CM was collected and added toastrocytoma U-87 cells The U-87 cells were grown for 72 hafter which the CM was spun and concentrated six times usingan Amicon device The cells were washed and suspended inbuffer and CE and CM were analyzed by WB in a 12 PAAGγ-Synuclein-specific Ab 1H10D2 was used as the primary AbIsolation of Exosomes by High-Speed Centrifugation

The stable SH-SY5Y clones overexpressing γ-synuclein or γ-synuclein-GFP were split in a six-well plate in the regularmedium One day after the cells had reached 75minus80 of thedensity the medium was replaced with SFM (Invitrogen) After48 h the medium was collected into the sterile Eppendorf tubesand centrifuged for 15 min at 2000g The supernatant wascentrifuged for 20 min at 12000g the pellet was discarded andthe supernatant was subjected to ultracentrifugation (125000gfor 70 min at 4 degC) The pellets were washed with cold PBS

and suspended in 10 μL of hot BD buffer and then an equalvolume of 2times SB containing β-mercaptoethanol (ME) wasadded The samples were boiled and loaded onto a 12 PAAGThe cells from the same wells were also washed with cold PBSand suspended in hot BD buffer boiled sonicated andmeasured for their protein content using the BCA-1 proteinassay kit (Sigma St Louis MO)

Isolation of Exosomes Using ExoQuick PrecipitationSH-SY5Y cells overexpressing γ-synuclein were grown in theregular medium for 24 h the medium was replaced with SFMand growth was continued for 48 h CM was collected and spun(3000g for 10 min) to remove cells and cellular debrisExosomes were precipitated from CM by ExoQuick (SystemBiosciences Mountain View CA) as follows CM wasincubated overnight at 40 degC with ExoQuick in a 12 ratioAfter 24 h the mixture was centrifuged at 13000g for 30 minand both the supernatant and the pellet were analyzed by WB

Treatment with Inhibitors of Exosome BiogenesisTwo hours before the media was replaced with SFM methyl-β-cyclodextrin (Sigma) was added directly to the medium to afinal concentration of 25 μgmL A cell-permeable inhibitor ofneutral sphingomyelinase GW4869 (Sigma) was dissolved inDMSO and added for 48 h to SFM to a final concentration of75 μM The same amount of DMSO was added to controlwells

Western Blotting The protein samples were analyzed byWB in a 12 PAAG in the presence of SDS as describedpreviously4142 30 μg of total protein was loaded on each wellAfter electrophoresis proteins were transferred onto anImmobilon-FL transfer membrane with a pore size of 045μm (Millipore Chelmsford MA) Nonspecific binding siteswere blocked by immersing the membrane in Tris-bufferedsaline with 005 Tween 20 (TBST) and 10 nonfat dry milkfor 1 h at room temperature on an orbital shaker Forquantitative imaging of films Kodak Image Station 440 CF(Eastman Kodak Co Rochester NY) and Kodak DigitalScience Image Analysis were used Other details have beendescribed previously41minus44

In Vitro Mutagenesis Met38 and Tyr39 in recombinantproteins were substituted for Ala using a StratageneAgilentTechnologies Inc (Santa Clara CA) Quick Change Site-Directed Mutagenesis Kit (catalog no 200518) following theprocedures recommended by the manufacturer For thereplacement of Met38 with Ala oligonucleotide 5prime-CCA AGGAGG GGG TCG CGT ATG TGG GAG CCA-3prime was used forthe replacement of Tyr39 with Ala we used oligonucleotide 5prime-AAG ACC AAG GAG GGG GTC ATG GCA GTG GGAGCC AAG ACC-3prime The complement oligonucleotide was 5prime-GGT CTT GGC TCC CAC TGC CAT GAC CCC CTCCTT GGT CTT-3prime For the replacement of both Met38 andTyr39 with Ala we used oligonucleotide 5prime-AAG ACC AAGGAG GGG GTC GCG GCA GTG GGA GCC AAG ACC-3primeand as a complement oligonucleotide 5prime-GGT CTT GGC TCCCAC TGC CGC GAC CCC CTC CTT GGT CTT-3prime Otherdetails of the procedure have been described previously43

cDNA pTrc-His-γ-synuclein was used as a templateGeneration of Recombinant γ-Synuclein Recombinant

proteins were expressed in E coli using cDNA pTrc-His-γ-synuclein in the presence of 1 mM IPTG as an inducer ofexpression Proteins were purified using a ProBond (Invi-trogenLife Technologies Corp Grand Island NY) columnfollowing the instructions for ProBond column users Purifiedproteins were at least 98 pure



TEM of γ-Synuclein Aggregates γ-Synuclein wasincubated with or without DA After being incubated overnightat 37 degC the samples were applied to 300 mesh Formvarcarbon-coated grids and negatively stained with 1 uranylacetate A JEOL 100CXII TEM instrument was used toexamine the samples Images were photographed at a

magnification of 36000times on Kodak electron image filmNegatives were developed and scanned into TIF files usingan Epson Perfection 4990 photo scanner

Mass Spectrometry The mass spectra of the undigestedprotein were recorded using a Q-TOF-2 (Micromass LtdManchester UK) mass spectrometer CapLC-LTQ-FT-MS

Figure 1 Aggregation of α- β- and γ-synucleins in vitro and in cell cultures (A) Schematic drawing of α- β- and γ-synucleins (B) Synucleinaggregation in vitro Samples 1 4 and 7 were incubated at 4 degC samples 2 5 and 8 at 37 degC and samples 3 6 and 9 at 37 degC with 1 mM DA WBwas conducted with Ab to α-synuclein (lanes 1minus3) β-synuclein (lanes 4minus6) and γ-synuclein (lanes 7minus9) Significant amounts of aggregated α- andγ-synucleins were formed after overnight incubation of proteins at 37 degC in the presence of DA (lanes 3 and 9 between arrowheads) α- and β-synuclein monomers (arrow) were converted to aggregates only partially (lanes 3 and 6) whereas γ-synuclein was almost completely turned intoaggregates (lane 9) (C) Oxidized wt-γ-synuclein induces aggregation of α-synuclein γ-Synuclein was oxidized separated from DA and incubatedovernight with α-synuclein The samples were subjected to WB with α-synuclein-specific Ab lane 1 α-synuclein incubated with oxidized γ-synucleinin a 51 ratio lane 2 same as lane 1 but with a 201 ratio lane 3 α-synuclein incubated with untreated γ-synuclein in a 51 ratio lane 4 same as lane3 but with a 201 ratio The formation of both α-synuclein dimers and aggregates (sim250minus300 kDa) was observed only when α-synuclein wasincubated with oxidized γ-synuclein in a 51 ratio (lane 1 arrowhead) (D) Oxidized γ-synuclein forms a heteromeric complex in vitro with α-synuclein but not β-synuclein γ-Synuclein was treated with DA and incubated with α-synuclein (lanes 2 3 8 and 9) or β-synucleins (lanes 5 6 10and 11) DA was eliminated by dialysis in samples 2 5 8 and 10 WB was performed using Ab to α-synuclein (lanes 1minus3) β-synuclein (lanes 4minus6)and γ-synuclein (lanes 7minus11) Lane 7 contained untreated γ-synuclein γ-Synuclein nucleates aggregation of α-synuclein but not β-synuclein High-molecular mass aggregates (sim250minus300 kDa arrowhead) contain both α-synuclein (lanes 2 and 3) and γ-synuclein (lanes 8 and 9)

Figure 2 Mass spectrometry analysis of γ-synuclein (A and B) Q-TOF spectra of the whole (undigested) γ-synuclein incubated with either buffer(A) or DA (B) An increase of 32 Da was found for the DA-treated protein sample (C and D) Representative capLC-LTQ-FT-MSMS spectrademonstrating the presence of oxidized (mass increase of 16 Da) Met38 (C) or Tyr39 (D) in the γ-synuclein tryptic peptide sequenceTKEGVM38Y39VGAK after DA treatment



MS spectra for in-solution or in-gel protein digests wererecorded on an LTQ-FT instrument (ThermoElectron SanJose CA) as described previously45 Additionally raw data wereexamined manually using Mass Matrix (httpwwwmassmatrixnet)Inhibition of Proteasome Activity SH-SY5Y cells were

grown for 24 h in the regular medium with subsequentreplacement of the medium with SFM Cells were treated withproteasome inhibitor MG132 (10 μM for 24 h) CM washarvested centrifuged (10 min at 3000g) and concentrated 10times using the Amicon device with membrane-retainingproteins with a molecular mass of gt10 kDaStatistical Analysis At least three sets of WB were

performed for each experiment A Studentrsquos t test was used toassess significant differences between groups The effect ofinhibitors was analyzed by ANOVA

RESULTSAggregation of Synucleins in Vitro Incubation of

synucleins at 37 degC is accompanied by a slow oligomerizationand aggregation DA significantly accelerates the level ofsynuclein oligomerization producing protein complexes withmolecular masses of sim250 kDa (in Figure 1B aggregates areseen in lanes 3 6 and 9 between arrowheads) Importantly γ-synuclein most easily forms high-molecular mass aggregatescompared to two other members of the family Almost all γ-synuclein monomer (arrow) was converted to oligomeric form(Figure 1B lane 9 between arrowheads) whereas theconversion of α- and β-synuclein monomers to aggregatedspecies was only partial (Figure 1B lanes 3 and 6 arrow)Furthermore oxidation of γ-synuclein not only causes itsaggregation but also induces its ability to seed the aggregationof α-synuclein (Figure 1C lane 1 arrowhead Figure 1D lanes2 3 8 and 9) Aggregation of β-synuclein was not affected(Figure 1D lanes 5 and 6) At a higher α-synucleinγ-synucleinratio (201) (Figure 1C lane 2) or after incubation of α-synuclein with untreated γ-synuclein (Figure 1C lanes 3 and4) α-synuclein aggregates were not found Because γ-synuclein-induced α-synuclein aggregates (sim250 kDa) are γ-synucleinimmunopositive (Figure 1D lanes 9minus11) they can beconsidered as heterooligomers containing both these proteinsAnalysis of DA-Treated γ-Synuclein by Mass Spec-

trometry We then sought to determine whether the increasedaggregation propensity of γ-synuclein after DA treatment is dueto a chemical modification of the protein Analysis of massspectra of the whole (undigested) protein revealed that afterDA treatment most of the protein (sim90) was converted tothe doubly oxidized isoform (addition of 32 Da to the originalmass of 13332 Da) Approximately 10 of the sample wasrepresented by an intact protein while no monooxidizedisoform was found (Figure 2AB) Tandem MS analysis of γ-synuclein tryptic digests after DA treatment showed thepresence of oxidized Met38 and Tyr39 (panels C and D ofFigure 2 respectively) No other oxidation modifications havebeen detected at sim70 sequence coverage The MSMSspectra for the two adjacent oxidation sites are very similarhave exactly the same ion precursor mass and can bedistinguished only through a difference in y5 (5373 vs5533) and b4 (4152 vs 3992) fragment ion mz values forMet38(O) and Tyr39(O)Role of Met38 and Tyr39 in the Propensity of γ-

Synuclein To Aggregate We then asked whether theoxidation of Met38 and Tyr39 might play a role in the

susceptibility of γ-synuclein to aggregation in vitro For thispurpose we replaced these two residues with alanine andcompared the propensity of wt and mutant forms of γ-synucleinto aggregate After incubation with DA wt-γ-synuclein formshigh-molecular mass aggregates that can be detected byCoomassie staining (Figure 3A lane 3 arrowhead) Similar

size aggregates were revealed by WB (Figure 1B lane 9)however only the most abundant forms of aggregates wererevealed by Coomassie staining because of the lower sensitivityof this method of detection The substitution of Met38 with Alareduced the level of aggregation of the protein (lane 4untreated lane 5 DA-treated M38A γ-synuclein) the majorityof which remained in the form of a monomer (Figure 3Aarrow) Similar results were obtained for γ-synuclein in whichTyr39 had been substituted with Ala (not shown) Thus thepropensity for γ-synuclein to aggregate under oxidativeconditions in vitro depends on the presence of Met38 andTyr39 in adjacent positions The role of these amino acids in thestructural organization and fibrillation of α-synuclein4647 and inmodel peptides48 has been demonstrated previously Thepresence of the Met38-oxidized form of γ-synuclein in thefraction of aggregated protein was confirmed by the use of Abraised to γ-synuclein with oxidized Met38 (Figure 3B lane 2arrowhead) This Ab reveals only minor amounts of aggregatesin the recombinant γ-synuclein with the Met38 rarr Alasubstitution (Figure 3B lane 4)

Analysis of Synuclein Oligomers by TEM According toTEM γ-synuclein oligomers generated after oxidative treatmenthave a ring- or horseshoelike shape with a size of 30minus50 nm(Figure 4ABD) and look like the annular oligomers describedpreviously for α-synuclein4950 Similar but less numerousannular oligomers are formed upon incubation of γ-synuclein in

Figure 3 Role of Met38 in the propensity of γ-synuclein to aggregate(A) Substitution of Met38 reduces the propensity of γ-synuclein toaggregate The recombinant protein with Met38 substituted for Ala wasincubated with DA (lanes 3 and 5) or buffer (lanes 2 and 4) AfterPAGE the gel was stained with Colloidal Coomassie Brilliant BlueAlmost all wt-γ-synuclein forms high-molecular mass aggregates (lane3 arrowhead) whereas a mutant with the Met38 rarrAla substitutionremains basically monomeric (lanes 4 and 5 arrow) lane 1 molecularmass markers lane 2 wt-γ-synuclein incubated without DA lane 3 wt-γ-synuclein incubated with DA lane 4 γ-synuclein with the Met38 rarrAla substitution incubated with buffer lane 5 γ-synuclein with theMet38 rarr Ala substitution incubated with DA (B) The Ab raised to γ-synuclein with oxidized Met38 reveals a high-molecular mass form of γ-synuclein (lane 2 arrowhead) Such aggregates are absent in untreatedγ-synuclein with the Met38 rarr Ala substitution (lane 3) and innonoxidized wt-γ-synuclein (lane 1) wt wild-type γ-synuclein mMet38 rarr Ala mutant + incubation with DA minus incubation withbuffer



the absence of DA (Figure 4C) α-Synuclein aggregatesgenerated in the presence of oxidized γ-synuclein have eithera rodlike (Figure 4E aminush) or an annular (Figure 4E iminusk)shape (sim200 nm length for rodlike and sim70 nm diameter forannular oligomers)γ-Synuclein Aggregation in Neuronal Cells and

Astrocytes Next we examined localization and aggregationof oxidized γ-synuclein in different types of cultured cellsAccording to immunofluorescence microscopy oxidized γ-synuclein has a perinuclear punctuate appearance and mayaccumulate in neuronal SH-SY5Y cells in the form ofintracellular inclusions (Figure 5A panels b and d) whereasmonomeric γ-synuclein is located predominantly in thecytoplasm and does not form deposits (Figure 5A panels aand c) Addition of the oxidized oligomeric γ-synuclein toastrocytes causes its internalization and accumulation inside thecells in its aggregated form (Figure 5B lane 6) with a molecularmass of sim250 kDaThen we sought to determine whether the induction of α-

synuclein aggregation by γ-synuclein in vitro might occur incells To reveal whether γ-synuclein affects α-synucleinaggregation in neuronal cells we used two approaches for thedelivery of γ-synuclein into SH-SY5Y cells (1) overexpressionusing the pEGFP-N1 plasmid containing human wild-type andmutant (Met38 rarr Ala) γ-synuclein cDNA (Figure 5CE) and(2) protein delivery agent BioPorter (Figure 5DF) Aggregatedα-synuclein was identified with the Ab against α-synucleinphosphorylated at Ser129 [anti-α-synuclein (pSer129) rabbitpAb] because α-synuclein accumulates in cell cultures inhyperphosphorylated states recapitulating key features ofintracellular inclusions in PD21 As shown in lane 3 of Figure5C and in Figure 5E the amount of aggregated α-synuclein(molecular mass of sim200 kDa) was significantly augmented(56-fold increase n = 3) when the cells were transfected withwt-γ-synuclein while mutant γ-synuclein (Met38 rarr Ala

substitution lane 2) caused a less significant increase (36-fold n = 3) A similar effect was observed when either intact γ-synuclein or DA-treated γ-synuclein was delivered by BioPorter(lane 2 or 3 of Figure 5D respectively and Figure 5F) Thus γ-synuclein delivered either by transfection or by protein deliveryagent enhanced the aggregation of α-synuclein in neuronalcells

Secretion and Internalization of γ-Synuclein Accordingto the literature data α-synuclein can be secreted from cells inthe form of exosomes30 We asked whether secretion andinternalization of γ-synuclein occur through a similarmechanism To answer this question we first overexpressedγ-synuclein in the neuronal SH-SY5Y cell culture with andwithout the GFP tag and analyzed the conditioned medium(CM) and cell extracts (CE) by WB As shown in Figure 6Aboth monomeric γ-synuclein and the γ-synuclein-GFP fusionprotein are secreted into CM γ-Synuclein expressed withoutthe GFP tag is secreted predominantly as a monomer (Figure6A lane 4) with a size corresponding to the size of theintracellular protein (Figure 6A lane 1) Addition ofproteasome inhibitor MG132 does not affect the size of thesecreted protein (not shown) γ-Synuclein fused to GFPmigrates in a PAAG according to the sum of the molecularmasses of two proteins (sim42 kDa) as seen in lanes 2 3 5 and6We then asked whether γ-synuclein secreted from a neuronal

culture can be internalized by astrocytoma cells SH-SY5Ystable clones overexpressing either γ-synuclein or γ-synuclein-pGFP were grown in SFM for 48 h CM was collected filteredthrough a 02 μm filter and added to astrocytoma U-87 cellswhich were split in the regular medium a day before Figure 6Bshows the uptake of extracellular γ-synuclein by astrocytomacells The majority of γ-synuclein in astrocytoma CE is presentas low-molecular mass aggregates (35minus60 kDa lanes 7 and 8)whereas in CM higher-molecular mass aggregates prevail (40minus

Figure 4 Analysis of α- and γ-synuclein aggregates by TEM γ-Synuclein was incubated overnight at 37 degC with DA (A and B) or without DA (C) asdescribed in the legend of Figure 1 The samples were applied to 300 mesh Formvarcarbon-coated grids and negatively stained with 1 uranylacetate (A and B) Random fields of γ-synuclein incubated with DA (C) γ-Synuclein incubated without DA (D) Selected round aggregates (E)TEM micrographs illustrating α-synuclein prefibrillar oligomers formed in the presence of oxidized γ-synuclein (51 ratio) The oligomers have eithera rodlike (aminush) or an annular (iminusk) shape (sim200 nm length for rodlike and sim70 nm diameter for annular oligomers) (l) α-Synuclein incubatedwithout γ-synuclein Met38 rarr Ala substitution



Figure 5 Internalization of γ-synucleins in glial and neuronal cells (A)Immunofluorescent staining of neuroblastoma SH-SY5Y cells revealedthe localization of monomeric and aggregated forms of γ-synuclein(red) Oxidized (b and d) and nonoxidized (a and c) forms of γ-synuclein were delivered by a cationic liposome reagent (BioPorterSigma) Conjugated PHA-L (Molecular Probes) labeled with Alexa488was used as a plasma membrane marker (green a and c) The Abagainst GM-130 (Becton Dickinson) was used as a Golgi marker(green b and d) DAPI denotes blue staining Only oxidized γ-synuclein forms intracellular deposits (red b and d arrows) which arecolocalized neither with the plasma membrane nor with Golgi markersThe data shown are representative of four independent experimentswith similar results (B) Addition of the aggregated γ-synuclein to A7astrocytes causes its intracellular accumulation γ-Synuclein wasincubated overnight in PBS buffer (lanes 1minus4) or in PBS with DA(lanes 5minus8) and then added to cultured A7 cells Cell extracts weresubjected to PAGE in a gradient 4 to 15 gel and analyzed by WBwith γ-synuclein Ab Cells were incubated with γ-synuclein as followslanes 1 and 5 no incubation lanes 2 and 6 incubation for 6 h lanes 3and 7 incubation for 24 h lanes 4 and 8 incubation for 48 hIntracellular γ-synuclein aggregates accumulated after a 6 h incubation(C) Overexpressed γ-synuclein enhances α-synuclein aggregation incultured SH-SY5Y cells infected with a lentivirus expressing α-synuclein lane 1 control clone transfected with an empty vector lanes2 and 3 stable clones overexpressing mutant Met38 rarr Ala γ-synuclein-GFP (lane 2) and wt-γ-synuclein-GFP (lane 3) The blot was probedwith the Ab specific to α-synuclein (bottom) stripped and probedwith the Ab to Ser129-phosphorylated α-synuclein (Wako) thatrecognizes predominantly aggregated α-synuclein21 (top) In thepresence of γ-synuclein the amount of aggregated α-synuclein(molecular mass of sim200 kDa) was increased 36 times by Met38 rarrAla γ-synuclein-GFP (lane 2) and 56 times by wt-γ-synuclein (lane 3)(n = 3) (D) γ-Synuclein delivered by BioPorter enhances α-synucleinaggregation lane 1 FITC-IgG delivered as a control lane 2 intact γ-synuclein lane 3 DA-treated γ-synuclein delivered with BioPorterThe Western blot was probed with the α-synuclein Ab for detection ofmonomeric α-synuclein (bottom) stripped and reprobed with the Abspecific to Ser129-phosphorylated α-synuclein Both intact (lane 2) andoxidized (lane 3) γ-synuclein delivered by a protein delivery agentincreased the level of α-synuclein aggregation (31 and 34 timesrespectively) compared to control (lane 1) (n = 3) (E and F) Extentsof induction of aggregated α-synuclein levels vs control (without γ-synuclein) presented as a bar graph corresponding to panels C and Drespectively were calculated on the basis of densities of bands fromthree independent experiments Asterisks show a statisticallysignificant difference (p lt 005)

Figure 6 Secretion of γ-synuclein from neuronal cells in the form ofexosomes and its transmission into astrocytes (A) Three types of γ-synuclein overexpressing stable SH-SY5Y clones were generated Inclone 1 γ-synuclein was expressed in the pcINeo vector (lane 1) inclone 2 γ-synuclein was fused with GFP (lane 2) and in clone 3mutant γ-synuclein with the Met38 rarr Ala substitution was fused withGFP (lane 3) Lane 4 contained CM from cells overexpressing the γ-synuclein monomer Lane 5 contained CM from cells overexpressingγ-synuclein-GFP Lane 6 contained CM from cells overexpressingmutant γ-synuclein-GFP The Western blot was probed with the γ-synuclein-specific Ab The molecular masses of marker proteins areshown at the left (B) γ-Synuclein in astrocytoma U-87 cells and CMCM medium from SH-SY5Y cells overexpressing γ-synuclein wasadded to U-87 cells After the cells had been grown for 72 h the CEand CM were analyzed by WB Lanes 1minus4 contained CM lanes 5minus8contained the corresponding CE lanes 1 and 5 U-87 control samplesincubated with fresh SFM lanes 2 and 6 samples from γ-synucleinstable clones overexpressing nonfused γ-synuclein lanes 3 and 7samples from the γ-synuclein-pGFP clone lanes 4 and 8 samples fromclones expressing mutant (Met38 rarr Ala) γ-synuclein-pGFP (C)Exosome samples were precipitated with ExoQuick and subjected toelectrophoresis in a 12 PAAG γ-Synuclein fused with GFP is presentin the exosomal pellet (P) precipitated from CM (lane 3) as a proteinwith a molecular mass of sim42 kDa that corresponds to the sum of themolecular masses of two proteins In the CM supernatant this band isless abundant (C lane 4) (D) The blot was stripped andimmunoblotted with the Ab to exosome marker CD63 (Santa CruzBiotechnology Inc 1100 dilution) lanes 1 and 2 CM from controlSH-SY5Y cells lanes 3 and 4 CM from cells overexpressing γ-synuclein-pGFP lanes 1 and 4 supernatant lanes 2 and 3 pellets afterthe precipitation of exosomes (E) Inhibitors of exosome biogenesisGW4869 and MBC reduce the level of secretion of monomeric γ-synuclein (m) and γ-synuclein fused to GFP (g-Syn-GFP) from SH-SY5Y cells lane 1 control sample for monomeric γ-synucleinincubated without inhibitors lane 4 control sample for the γ-synuclein-GFP-fused protein incubated without inhibitors GW4869caused a 58 plusmn 5 inhibition (n = 3) of γ-synuclein-GFP secretion (inpanel E compare lanes 4 and 5) MBC caused a significant inhibition(92 plusmn 4 n = 3) of secretion of the fused protein (in panel Ecompare lanes 4 and 6) Inhibition of the secretion of monomeric γ-synuclein by both inhibitors was less substantial compared to theinhibition of the fused protein MBC reduced the level of secretion ofmonomeric (m) γ-synuclein by 34 plusmn 6 (n = 3 compare lanes 1 and3) No statistically significant inhibition was found for GW4869 (Elane 2) (F) The amount of secreted exosome marker CD63 wasreduced in the presence of both inhibitors The average reduction inthe level of CD63 by GW4869 is 462 plusmn 6 (combined lanes 2 and 5compared to combined lanes 1 and 4 n = 3) the average reduction byMBC was 736 plusmn 4 (combined lanes 3 and 6 compared to combinedlanes 1 and 4 n = 3)



200 kDa lanes 3 and 4) Thus γ-synuclein can be internalizedby astrocytoma cells Interestingly nonfused γ-synuclein ispresent mainly as a monomer and a small fraction as a dimer inCM (Figure 6B lane 2) However in astrocytoma cells the CEmain band is a dimer and only traces of monomeric γ-synucleinare present (Figure 6B lane 6)Then we determined whether exosomes are implicated in γ-

synuclein secretion For this purpose we used two methods ofexosome isolation (a) a polymer-based method of exosomeprecipitation with ExoQuick and (b) a differential centrifuga-tion described previously30

First we precipitated exosomes with ExoQuick and analyzedthe presence of γ-synuclein in the exosome pellet (P) with theAb to γ-synuclein (Figure 6C) and exosomal marker CD63(Figure 6D) γ-Synuclein fused with GFP is present in theexosomal pellet precipitated from CM (Figure 6C lane 3) andin a considerably smaller amount in the CM supernatant (S)(Figure 6C lane 4) γ-Synuclein-GFP migrates in a PAAGaccording to the sum of the molecular masses of two proteins(42 kDa) A negligible amount of this protein was found incontrol SH-SY5Y cells that did not overexpress γ-synuclein(Figure 6C lanes 1 and 2) When the blot was stripped andincubated with the Ab to exosome marker CD63 the majorityof the antigen was present in the pellet (Figure 6D lanes 2 and3) but not in the supernatant (Figure 6D lane 1 and 4)Nonfused γ-synuclein also was localized in the exosome pelletas a protein with a molecular mass of sim14 kDa (not shown)Effect of Methyl-β-cyclodextrin (MBC) and GW4869

on the Secretion of γ-Synuclein Next we sought todetermine how inhibitors of exosome biogenesis affectsecretion of the monomeric γ-synuclein (designated with anldquomrdquo in Figure 6E) and γ-synuclein fused to GFP (gSyn-GFP inFigure 6E) We assessed the effect of two substances thatinhibit the formation of exosome components ie cholesteroland ceramide MBC is a cholesterol-depleting drug whileGW4869 is an inhibitor of neutral sphingomyelinase 2 anenzyme implicated in ceramide biosynthesis51minus53

The secretion of γ-synuclein-GFP from SH-SY5Y cells (lanes4minus6) is significantly inhibited by both GW4869 [58 plusmn 5inhibition n = 3 (Figure 6E lane 5)] and MBC [92 plusmn 5inhibition n = 3 (Figure 6E lane 6)] compared to the controlwithout inhibitors (lane 4) MBC inhibited secretion of the γ-synuclein monomer by 34 plusmn 5 (n = 3 lane 3) whereas noeffect of GW4869 on the secretion of monomer γ-synuclein wasfound (Figure 6E lane 2) The level of inhibition was moresignificant for γ-synuclein-GFP than for γ-synuclein without thetag The amount of exosome marker CD63 was also reduced inthe presence of both inhibitors (Figure 6 F) The averagereduction in the level of CD63 by GW4869 was 462 plusmn 5 (n =3 lane 5) and the average reduction caused by MBC was 736plusmn 6 (n = 3 lane 6)Intracellular Localization of the Secreted γ-Synuclein

in Astrocytoma Cells After U-87 astrocytoma cells had beengrown in the presence of CM from SH-SY5Y stable clonesoverexpressing γ-synuclein we observed the transmission of γ-synuclein into the cytoplasmic (Figure 7DF) and nuclear(Figure 7AC) compartments (arrows) PHA-L-lectin islocalized in the Golgi compartment (Figure 7B arrowhead)After 7 days γ-synuclein is localized in the nucleus (Figure7AC arrows) and cytoplasm (Figure 7DF arrows) If the cellsare double stained before fixation the lectin is localizedexclusively in the cell membranes and no γ-synuclein staining isobserved pointing to intracellular localization of γ-synuclein

After the culture had been stained for 12 days the formation offibrils localized mainly along the cell could be seen (Figure7GI double arrows) Without the primary Ab there is nostaining (not shown) The results of confocal microscopy withthe subsequent analysis of z-stacks also suggest nuclearlocalization of γ-synuclein (Figure 7N) confirming the datapresented in panels A and C of Figure 7

DISCUSSIONα-Synuclein has been linked to the pathogenesis of PD andother synucleinopathies through its propensity to form toxicaggregates but the upstream events causing α-synucleinaggregation are not completely understoodWe demonstrate here for the first time that the propensity of

α-synuclein to aggregate is enhanced by oxidized γ-synuclein(Figure 1) This property of γ-synuclein may play an importantrole in pathology because these two proteins are colocalized inseveral parts of the brain ie hippocampus amygdalebrainstem striatum and frontal cortex54 Both α-synucleinand γ-synuclein are expressed in motoneurons peripheralsensory neurons55 primary raphae nuclei neurons56 and othercell types The existence of heteromeric proteinminusproteincomplexes between γ-synuclein and α-synuclein that we

Figure 7 Fluorescence microscopy of astrocytoma U-87 cells (A DG and K) cells stained with γ-synuclein anti-rabbit polyclonal antibody(1250) and anti-rabbit TRITC (1150) as the secondary antibody(red) (B E H and L) PHA-L conjugated with Alexa488 (green)(1150) and (C F I and M) double staining by both dyes U-87 cellswere grown for 7 days (AminusF) and 12 days (GminusI) (KminusM) The cellswere stained with PHA-L conjugated with Alexa488 and γ-synucleinanti-rabbit polyclonal antibody before fixation Astrocytoma cells weregrown in the presence of CM taken from SH-SY5Y neuronal culturesγ-Synuclein is localized in the cytoplasm (D) and nucleus (A and Carrow) PHL-lectin is confined to the Golgi compartment (Barrowhead) After 12 days the formation of fibrils localized mainlyalong the long axis of the cell can be observed (G double arrow)Without the primary antibody there is no staining (not shown) If thecells were double stained before fixation the lectin was localizedexclusively in the cell membranes and no γ-synuclein staining wasobserved pointing to intracellular localization of γ-synuclein Theresults of confocal microscopy with the analysis of z-stacks support theintracellular localization of γ-synuclein part of which is confined to thenucleus (N)



demonstrate here by WB (Figure 1F) was revealed previouslyby co-immunoprecipitation56 The presence of heterodimerssuggests that the interaction between several members of thisfamily may play an essential role in the regulation of theiraggregational status Interestingly unmodified γ-synuclein andβ-synuclein possess chaperonic activity4257 and can inhibit α-synuclein fibrillation58minus60 so oxidative stress and post-transla-tional modifications significantly change synucleinrsquos functionalpropertiesThe body of evidence that pathological interactions among

different proteins that are prone to aggregate play an essentialrole in the formation of neurotoxic oligomers that accumulatein neurons or glial cells is growing Earlier the interactionbetween different naturally unfolded proteins has beendescribed for amyloid β (Aβ) and α-synuclein61minus63 tau andα-synuclein64 and Aβ and tau65 The broader implications ofsuch hybrid interactions might be important in the patho-genesis of other protein misfolding diseasesA growing body of published evidence reveals the

considerable overlap of clinicopathological features amongNDDs This overlap can be explained by the formation ofhybrid misfolded protein oligomers61minus66 Oxidative stress is animportant contributing factor in the pathogenesis of manyNDDs The level of oxidatively modified proteins in PD andsynucleinopathies is increased leading to the impairment ofseveral cellular functions6768

We show here that γ-synuclein is the most easily oxidizedmember of the synuclein family and that it forms aggregatesafter oxidation Aggregates can be detected by Coomassiestaining (Figure 3A lane 3) and WB (Figure 1B lane 9)Importantly oxidized γ-synuclein seeds α-synuclein aggregationboth in vitro (Figure 1CD) and in vivo (Figure 5CD) Easyoxidation of Met38 and Tyr39 in γ-synuclein may be explainedby the peculiarities of its amino acid sequence and secondarystructure In α-synuclein the corresponding positions areoccupied by Leu38 and Tyr39 (Figure 1A) located in a linkerbetween two α-helices69 γ-Synuclein has a similar molecularstructure except for the C-terminal part of the molecule (figurein the Table of Contents)70 The linker is a loosely packed andsurface-exposed region that is more readily accessible tooxygen71 Interestingly the linker between two helices islocated on the boundary between the first and second exonsand its amino acid sequence is conserved in human cowmouse rat porcine and Xenopus α-synuclein70 suggesting thatsuch a structural feature is preserved during evolutionLocalization of Met38 in a position proximal to Tyr39 allowsintramolecular oxygen transfer and enhances the propensity toaggregate48 Tyr39 plays a critical role in α-synucleinfibrillation1072 and its oxidative modification affects itsstructure and aggregational properties73Methionine residuesare important for the long-range interactions and secondarystructure of α-synuclein Furthermore oxidation of methionineresidues in α-synuclein affects electrostatic and hydrophobicinteractions and the propensity to fibrillate74

We have found that the substitution of Met38 or Tyr39 withAla reduces the level of aggregation of the protein Thus thepropensity of γ-synuclein to aggregate under oxidativeconditions in vitro depends on Met38 and Tyr39 localized inadjacent positions This structural property together with thepresence of a hydrophobic stretch of amino acids (NACdomain) in the central part of its molecule (Figure 1A)presumably facilitates α-synuclein aggregation and its ability toinduce the aggregation of α-synuclein

In another member of the synuclein family α-synucleinTyr39 plays a critical role in its fibrillation4648 while in proteinswith Tyr and Met in neighboring positions an efficientintramolecular oxygen transfer occurs to give a dioxygenatedderivative with one oxygen on the Tyr and the other formingmethionine sulfoxides In this case Tyr may play a catalyticalrole to produce Met oxidation48

The annular forms of oxidized γ-synuclein that we havedescribed here might have other deleterious effect on cellviability affecting membrane permeability as shown previouslyfor α-synuclein and other amyloid proteins4975minus77 Some α-synuclein aggregates seeded by γ-synuclein also have annularstructure (Figure 4E panels iminusk) suggesting that γ-synucleinoxidation may trigger the accumulation of porelike synucleinaggregates Synuclein-containing porelike structures within cellmembranes may cause a disruption of cellular ion homeostasisincluding channel-mediated calcium influx and subsequent celldeath6375minus77

Oxidized γ-synuclein accumulates in neuronal cells and formsintracellular inclusions (Figure 5AB) Furthermore γ-synucleincan be secreted from cells into CM in aggregated form (Figure6) In the presence of proteasome inhibitors higher-molecularmass aggregates are formed (Figure 6A) Aggregated γ-synuclein can be internalized by astrocytoma cells (Figure6D) This process is accompanied by the alteration of thepattern of aggregation which may be explained by changes inprotein conformation andor post-translational modificationsIn the presence of a cholesterol-depleting drug MBC and an

inhibitor of ceramide biosynthesis GW4869 the extent of γ-synuclein secretion as well as the amount of exosome markerCD63 was decreased (Figure 6E) further confirming that γ-synuclein export takes place through exosomesThe ability of γ-synuclein to seed α-synuclein aggregation

found in vitro (Figure 1) was reproduced in experiments withcell cultures (Figure 5) However when we analyzed the abilityof γ-synuclein to seed α-synuclein aggregation in vitro and incell cultures we revealed some differences For example DA-treated γ-synuclein seeded α-synuclein aggregation moreefficiently than the intact protein in vitro (Figure 1) whereasno differences were found in cell cultures (Figure 1E lanes 2and 3) These discrepancies may be explained by γ-synucleinpost-translational modifications that change its properties in thecellular milieuThe discovery of α-synuclein in extracellular biological fluids

including human CSF and blood plasma in both healthysubjects and patients with PD11minus13 suggests that extracellular α-synuclein may physiologically or pathologically affect bothneuronal and glial brain cells26Importantly the levels ofoligomeric α-synuclein in CSF and blood plasma are elevated inPD patients1213 Secretion of α-synuclein into extracellularfluids is an important first step in the neuron to neuron orneuron to glial cell propagation of pathology in NDDs162880

The data presented here show that γ-synuclein alsoparticipates in secretion and internalization It can be secretedinto CM and internalized by neuronal or glial cells resulting inthe intracellular accumulation and deposition of γ-synuclein Inaddition oxidized γ-synuclein seeds α-synuclein aggregationand therefore possesses prion-like properties Thus synucleinsexert their pathogenic actions not only in the cytoplasm ofneuronal cells that strongly express these proteins but also inthe extracellular space affecting the function of neighboringcells These results provide new insights into the events thatregulate the formation of intracellular aggregated α-synuclein



and further elucidate the pathological mechanisms underlyingPD and other synucleinopathiesRecent data suggest that under physiologic conditions α-

synuclein exists in a tetrameric form that has considerablehelical content7879 Our results indicate that γ-synuclein ispresent in strongly overexpressing cells as a SDS-resistanttetramer These new findings demonstrate that further studiesare required to improve our understanding of factors regulatingthe state of synuclein aggregation Among these factors arepost-translational modifications of synucleins and theirinteraction with other proteins as well as with small moleculesthat could affect the level of aggregation and change synucleinpathogenicityA better understanding of the steps involved in the process of

α-synuclein aggregation and the role of γ-synuclein as a coreinducing such aggregation is important for developingintervention strategies that would prevent or reverse theaccumulation of toxic proteins in NDDs

AUTHOR INFORMATION

Corresponding AuthorRetinal Biology Laboratory Veterans Administration MedicalCenter 4801 E Linwood Blvd Kansas City MO 66148Telephone (816) 861-4700 ext 57078 Fax (816) 861-1110E-mail asurguchovkumcedu

FundingThis work was supported by a VA Merit Review Grant and theGlaucoma Foundation

NotesThe authors declare no competing financial interest

ACKNOWLEDGMENTS

We thank Dr Herbert M Geller of the National Heart Lungand Blood Institute for the A7 astrocyte cell culture and DrElieser Masliah from the University of California San Diego(La Jolla CA) for the lentiviral constructs expressing wild-typeand A53T mutant α-synuclein We are grateful to ProfessorVirginia Savin for the critical reading of the manuscript We arealso grateful to Barbara Fegley for assistance with TEM

ABBREVIATIONS

Ab antibody CE cell extracts CM conditioned medium DAdopamine DMEM Dulbeccorsquos modified Eaglersquos mediumMBC methyl-β-cyclodextrin NDD neurodegenerative dis-eases PAAG polyacrylamide gel PAGE polyacrylamide gelelectrophoresis PBS phosphate-buffered saline PD Parkin-sonrsquos disease PFA paraformaldehyde RT room temperatureTEM transmission electron microscopy WB Westernblotting wt wild type

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astrocytes are constitutively phagocytic and have synuclein dependentreactivity in glaucoma Proc Natl Acad Sci USA 108 1176minus1181(40) Geller H M and Dubois-Dalcq M (1988) Antigenic andfunctional characterization of a rat central nervous system-derived cellline immortalized by a retroviral vector J Cell Biol 107 1977minus1986(41) Surgucheva I Park B C Yue B Y Tomarev S andSurguchov A (2005) Interaction of myocilin with γ-synuclein affectsits secretion and aggregation Cell Mol Neurobiol 25 1009minus1033(42) Surgucheva I Ninkina N Buchman V L Grasing K andSurguchov A (2005) Protein aggregation in retinal cells andapproaches to cell protection Cell Mol Neurobiol 25 1051minus1066(43) Surgucheva I and Surguchov A (2008) γ-Synuclein Cell-type-specific promoter activity and binding to transcription factors J MolNeurosci 35 267minus271(44) Surguchov A Palazzo R E and Surgucheva I (2001) γ-Synuclein Subcellular localization in neuronal and non-neuronal cellsand effect on signal transduction Cell Motil Cytoskeleton 49 218minus228(45) Sharov V S Galeva N A Dremina E S Williams T D andSchoneich C (2009) Inactivation of rabbit muscle glycogenphosphorylase b by peroxynitrite revisited Does the nitration ofTyr613 in the allosteric inhibition site control enzymatic functionArch Biochem Biophys 484 155minus166(46) Ulrih N P Barry C H and Fink A L (2008) Impact of Tyrto Ala mutations on α-synuclein fibrillation and structural propertiesBiochim Biophys Acta 1782 581minus585(47) Lamberto G R Binolfi A Orcellet M L Bertoncini C WZweckstetter M Griesinger C and Fernandez C O (2009)Structural and mechanistic basis behind the inhibitory interaction ofPcTS on α-synuclein amyloid fibril formation Proc Natl Acad SciUSA 106 21057minus21062(48) Nagy P Kettle A J and Winterbourn C C (2009)Superoxide-mediated formation of tyrosine hydroperoxides andmethionine sulfoxide in peptides through radical addition andintramolecular oxygen transfer J Biol Chem 284 14723minus14733(49) Lashuel H A Petre B M Wall J Simon M Nowak R JWalz T and Lansbury P T Jr (2002) α-Synuclein especially theParkinsonrsquos disease-associated mutants forms pore-like annular andtubular protofibrils J Mol Biol 322 1089minus1102(50) Pountney D L Voelcker N H and Gai W P (2005)Annular α-synuclein oligomers are potentially toxic agents in α-synucleinopathy Hypothesis Neurotoxic Res 7 59minus67(51) Trajkovic K Hsu C Chiantia S Rajendran L Wenzel DWieland F Schwille P Brugger B and Simons M (2008)Ceramide triggers budding of exosome vesicles into multivesicularendosomes Science 319 1244minus1247(52) Bar-On P Rockenstein E Adame A Ho G Hashimoto Mand Masliah E (2006) Effects of the cholesterol-lowering compoundmethyl-β-cyclodextrin in models of α-synucleinopathy J Neurochem98 1032minus1045(53) Kosaka N Iguchi H Yoshioka Y Takeshita F Matsuki Yand Ochiya T (2010) Secretory mechanisms and intercellular transferof microRNAs in living cells J Biol Chem 285 17442minus17452(54) Jeannotte A M McCarthy J G and Sidhu A (2009)Desipramine induced changes in the norepinephrine transporter α-and γ-synuclein in the hippocampus amygdala and striatum NeurosciLett 467 86minus89(55) Ninkina N Papachroni K Robertson D C Schmidt ODelaney L OrsquoNeill F Court F Rosenthal A Fleetwood-Walker SM Davies A M and Buchman V L (2003) Neurons expressing thehighest levels of γ-synuclein are unaffected by targeted inactivation ofthe gene Mol Cell Biol 23 8233minus8245(56) Wersinger C and Sidhu A (2009) Partial regulation ofserotonin transporter function by γ-synuclein Neurosci Lett 453 157minus161(57) Jiang Y Liu Y E Goldberg I D and Shi Y E (2004) γ-Synuclein a novel heat-shock protein-associated chaperone stimulatesligand-dependent estrogen receptor α signaling and mammarytumorgenesis Cancer Res 64 4539minus4546



(58) Biere A L Wood S J Wypych J Steavenson S Jiang YAnafi D Jacobsen F W Jarosinski M A Wu G M Louis J CMartin F Narhi L O and Citron M (2000) Parkinsonrsquos disease-associated α-synuclein is more fibrillogenic than β- and γ-synuclein andcannot cross-seed its homologs J Biol Chem 275 34574minus34579(59) Hashimoto M Rockenstein E Mante M Mallory M andMasliah E (2001) β-Synuclein inhibits α-synuclein aggregation Apossible role as an anti-parkinsonian factor Neuron 32 213minus223(60) Uversky V N Li J Souillac P Millett I S Doniach S JakesR Goedert M and Fink A L (2002) Biophysical properties of thesynucleins and their propensities to fibrillate Inhibition of α-synucleinassembly by β- and γ-synucleins J Biol Chem 277 11970minus11978(61) Mandal P K Pettegrew J W Masliah E Hamilton R L andMandal R (2006) Interaction between Aβ peptide and α-synucleinMolecular mechanisms in overlapping pathology of Alzheimerrsquos andParkinsonrsquos in dementia with Lewy body disease Neurochem Res 311153minus1162(62) Masliah E Rockenstein E Veinbergs I Sagara Y MalloryM Hashimoto M and Mucke L (2001) β-Amyloid peptidesenhance α-synuclein accumulation and neuronal deficits in atransgenic mouse model linking Alzheimerrsquos disease and Parkinsonrsquosdisease Proc Natl Acad Sci USA 98 12245minus12250(63) Tsigelny I F Crews L Desplats P Shaked G M SharikovY Mizuno H Spencer B Rockenstein E Trejo M Platoshyn OYuan J X and Masliah E (2008) Mechanisms of hybrid oligomerformation in the pathogenesis of combined Alzheimerrsquos andParkinsonrsquos diseases PLoS One 3 e3135(64) Giasson B I Forman M S Higuchi M Golbe L I GravesC L Kotzbauer P T Trojanowski J Q and Lee V M (2003)Initiation and synergistic fibrillization of tau and α-synuclein Science300 636minus640(65) Lewis J Dickson D W Lin W L Chisholm L Corral AJones G Yen S H Sahara N Skipper L Yager D Eckman CHardy J Hutton M and McGowan E (2001) Enhancedneurofibrillary degeneration in transgenic mice expressing mutanttau and APP Science 293 1487minus1491(66) Crews L Tsigelny I Hashimoto M and Masliah E (2009)Role of synucleins in Alzheimerrsquos disease Neurotoxic Res 16 306minus317(67) Butterfield D A Perluigi M and Sultana R (2006) Oxidativestress in Alzheimerrsquos disease brain New insights from redoxproteomics Eur J Pharmacol 545 39minus50(68) Przedborski S (2005) Pathogenesis of nigral cell death inParkinsonrsquos disease Parkinsonism Relat Disord 11 (Suppl 1) S3minusS7(69) Ulmer T S Bax A Cole N B and Nussbaum R L (2005)Structure and dynamics of micelle-bound human α-synuclein J BiolChem 280 9595minus9603(70) Manivel P Muthukumaran J Kannan M and Krishna R(2011) Insight into residues involved in the structure and function ofthe breast cancer associated protein human γ-synuclein J Mol Model17 251minus263(71) Chen M Margittai M Chen J and Langen R (2007)Investigation of α-synuclein fibril structure by site-directed spinlabeling J Biol Chem 282 24970minus24979(72) Lamberto G R Binolfi A Orcellet M L Bertoncini C WZweckstetter M Griesinger C and Fernandez C O (2009)Structural and mechanistic basis behind the inhibitory interaction ofPcTS on α-synuclein amyloid fibril formation Proc Natl Acad SciUSA 106 21057minus21062(73) Sevcsik E Trexler A J Dunn J M and Rhoades E (2011)Allostery in a disordered protein oxidative modifications to α-synuclein act distally to regulate membrane binding J Am Chem Soc133 7152minus7158(74) Zhou W Long C Reaney S H Di Monte D A Fink A Land Uversky V N (2010) Methionine oxidation stabilizes non-toxicoligomers of α-synuclein through strengthening the auto-inhibitoryintra-molecular long-range interactions Biochim Biophys Acta 1802322minus330

(75) Volles M J and Lansbury P T Jr (2002) Vesiclepermeabilization by protofibrillar α-synuclein is sensitive toParkinsonrsquos disease-linked mutations and occurs by a pore-likemechanism Biochemistry 41 4595minus4602(76) Tsigelny I F Sharikov Y Miller M A and Masliah E(2008) Mechanism of α-synuclein oligomerization and membraneinteraction Theoretical approach to unstructured protein studiesNanomedicine 4 350minus357(77) Tsigelny I F Bar-On P Sharikov Y Crews L HashimotoM Miller M A Keller S H Platoshyn O Yuan J X and MasliahE (2007) Dynamics of α-synuclein aggregation and inhibition of pore-like oligomer development by β-synuclein FEBS J 274 1862minus1877(78) Bartels T Choi J G and Selkoe D J (2011) α-Synucleinoccurs physiologically as a helically folded tetramer that resistsaggregation Nature 477 107minus110(79) Wang W Perovic I Chittuluru J Kaganovich A Nguyen LT Liao J Auclair J R Johnson D Landeru A Simorellis A K JuS Cookson M R Asturias F J Agar J N Webb B N Kang CRinge D Petsko G A Pochapsky T C and Hoang Q Q (2011) Asoluble α-synuclein construct forms a dynamic tetramer Proc NatlAcad Sci USA 108 17797minus17802(80) Dunning C J Reyes J F Steiner J A and Brundin P (2012)Can Parkinsonrsquos disease pathology be propagated from one neuron toanother Prog Neurobiol 97 205minus219



aberrant accumulation these disorders may be termed γ-synucleinopathies3338

Here we show that γ-synuclein can seed α-synucleinaggregation this seeding is especially efficient after γ-synucleinoxidation Furthermore γ-synuclein can be secreted fromneuronal cell cultures and internalized by glial cells Theseprocesses might have an important role in the development andspreading of synucleinopathies

EXPERIMENTAL PROCEDURES

Antibodies (Abs) for WB and ImmunofluorescenceThe following Abs were purchased from Santa CruzBiotechnology Inc (Santa Cruz CA) α-synuclein-specificantibody [sc-7011-R (C-20)] and CD63-specific Ab (H-193)(sc-15363) β-Synuclein anti-rabbit polyclonal Ab AB5068 wasobtained from Chemicon (Temecula CA) γ-Synuclein Absused for WB were either anti-rabbit (Ab6169) or anti-mouse(1H10D2) clones from ABcam (Cambridge MA) Mousemonoclonal Ab 2C3 against γ-synuclein (GenTex) was used forimmunofluorescence The Ser129-phosphorylated α-synucleinwas revealed with anti-mouse monoclonal Ab from Wacowhich recognizes predominantly aggregated α-synuclein21

Lectin-PHA-L-conjugated by Alexa488 (InvitrogenMolecularProbes Eugene OR) was used as a membrane marker and anti-mouse Ab against GM-130 [Becton Dickinson (BD) FranklinLakes NJ] as a Golgi marker Secondary Abs (goat anti-mouseAlexa488 and Qdot-655 anti-rabbit) were purchased fromInvitrogenMolecular Probes Anti-rabbit Cy3-conjugated Abwas from Jackson ImmunoResearch Lab Inc (West GrovePA)Generation of Ab to γ-Synuclein with an Oxidized

Methionine 38 (Met38) (21st Century BiochemicalsMarlboro MA) Ab was raised in rabbits using as an antigenpeptide Ac-TKEGV[M-O]YVGAKT-Ahx-C-amide where [M-O] is methionine sulfoxide The peptide was purified by high-performance liquid chromatography and its sequence wasconfirmed by MSMS Nanospray analysis After the fourthbleeding the Ab was affinity purified on a column containingantigenic peptide Purified Ab was immunodepleted using animmunodepletion column with unmodified peptide withoutoxidized methionine (acetyl-TKEGVMYVGAKT-Ahx-C-amide) For Western blotting (WB) the Ab was used at a1500 dilutionAnalysis of α- β- and γ-Synucleins Colloidal

Coomassie Brilliant Blue Staining The protein samples wereseparated in a 12 gel and fixed in 125 TCA for 1 h The gelwas stained with 01 G-250 brilliant blue in 20 methanolcontaining 10 (NH 4)2SO4 and 2 H3PO4 overnight at RTand washed with H2OAggregation Assays In Vitro Assay Recombinant α- β-

and γ-synucleins were from ABcam (α-synuclein ab48842 β-synuclein ab48853 and γ-synuclein ab48712 respectively) Theproteins were diluted by PBS to 05 μM mixed with a 20-foldexcess of DA and incubated overnight at 37 degC The sampleswere boiled for 3 min in a sample buffer (SB) with 5 β-mercaptoethanol After the treatment we assessed theformation of SDS-resistant protein aggregates by electro-phoresis in a 12 or 4 to 15 PAAGAssay of Cell Extracts A7 cells were split in a 96-well plate

in the regular medium The next day γ-synuclein preincubatedwith DA or PBS buffer was added to the cells The cells werewashed with PBS after different periods of incubation (0 6 24

and 48 h) and suspended in 2times SB The samples were boiledand analyzed in a 4 to 15 PAAG in the presence of SDS

Incubation with DA or L-DOPA α- β- and γ-synucleinswere incubated overnight with a 20-fold molar excess of DA inPBS Because of the low stability of DA in experiments withlonger periods of incubation it was replaced with a similarconcentration of a more stable DA precursor 34-dihydrox-yphenyl-L-alanine (L-DOPA)

Cell Cultures The immortalized primary culture of ratoptic nerve A7 astrocytes was a generous gift from H Geller(National Institutes of Health Bethesda MD)40 The culturewas immortalized with murine leukemia virus psi-2 SV-40-6which is defective in assembly and contains the SV-40 large Tantigen and neomycin-resistance genes as described previ-ously40 This stable immortalized clonal cell line expressednuclear SV-40 large T cells and the astrocyte-specific markerglial fibrillary acidic protein (GFAP) The cells were grown asdescribed previously41minus43

Human neuroblastoma cell line SH-SY5Y was purchasedfrom ATCC (catalog no CRL-2266) and cultivated in DMEMF-12 medium supplemented with 10 fetal bovine serum at 37degC in 5 CO2 Astrocytoma grade IVhigh-grade astrocytoma(U-87 MG) (ATCC catalog no HTB-14) was cultivated inEaglersquos Minimal Essential Medium with Earlrsquos BSS and 2 mMglutamine (EMEM) adjusted to contain 10 mM sodiumpyruvate 01 mM nonessential amino acids and 15 gLsodium bicarbonate (90) and fetal bovine serum (10)Other details are described by the manufacturer and in ourprevious publication42

Immunofluorescent Staining SH-SY5Y Cells They weresplit on coverslips covered with poly-D-lysine The cells werewashed with cold PBS and stained with PHA-L conjugated withAlexa488 for 15 min at 4 degC before being fixed (4 PFA for 15min at RT) After being fixed the cells were washed andblocked in 3 BSA 3 NGS and 01 Triton X-100 in PBSfor 2 h at RT The coverslips were incubated with the primaryAb overnight at 4 degC After being stained with the secondary Abfor 1 h at RT the coverslips were put in a mounting solutioncontaining DAPI-Vectashield (Vector Laboratories Burlin-game CA) Other details are described in ref 44

U-87 MG Cells They were split on the coverslips in a 12-wellplate in the regular medium The next day the medium wasreplaced for the CMs from the stable clones produced in SH-SY5Y cells The cells were grown for 7 or 12 days at 37 degC inthe presence of 5 CO2 Before being fixed the cells werewashed with warm PBS containing Ca2+ and Mg2+ and thenfixed with 4 PFA for 15 min at RT The cells were washedwith PBS permeabilized with 03 Triton X-100 (15 min atRT) and blocked in a solution containing 3 BSA 3 NGSand 01 Triton X-100 in PBS for 2 h at RT The coverslipswere placed in the primary antibody overnight at 4 degC Afterbeing stained with the secondary antibody for 1 h at RT thecoverslips were placed in a mounting solution containingDAPI-Vectashield (Vector Laboratories) Nonfixed cells werewashed with PBS and stained with PHA-L conjugated withAlexa488 for 2 h at RT before being fixed with 4 PFA for 15min at RT

Confocal Microscopy The images of U-87 cells weretaken with a Nikon TE 2000 Inverted 3 Laser confocalmicroscope driven by Nikon EZ-C1 391 software Thefollowing lasers were used 488 543 and 633 nm Nikon EZ-C1Free Viewer software was used for the reconstruction oforthogonal projections















































































AUTHOR INFORMATION




ACKNOWLEDGMENTS


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AUTHOR INFORMATION




ACKNOWLEDGMENTS


ABBREVIATIONS














γ-Synuclein: Seeding of α-Synuclein Aggregation and Transmission between Cells

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Transcript of γ-Synuclein: Seeding of α-Synuclein Aggregation and Transmission between Cells